Multiple in vitro mutagenesis reactions
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Wed Nov 17 04:20:54 EST 1999
In article <s33e4qmjhsq40 at corp.supernews.com>, Dr. David J. Meyer
<meyerdj at phibred.com> writes
>I have run 13 at a time before. In a small number of minipreps I found
>a couple which had 11/13 mutations.
That's very promising. What method did you use for the mutagenesis?
Plasmid or M13? Which polymerase and what selection, Kunkel?
Having thought again we are now thinking of screening not by
introduction or loss of RE sites but by making sure that we have a 'T'
change either in or next to the mutation we need. We will then screen by
T tracking on an automated sequencer, maybe on a PCR product. We should
be able to spot the mutants much quicker and easier. We are debating now
if we can multiplex PCRs i.e. Make as grid of 400 colonies in 20 rows by
20 columns. Run PCRs on pooled columns and pooled rows. That way we only
have to do 40 PCRs and 40 sequencing reactions. The question now is if
only one clone out of the 20 pooled ones has the mutation would we pick
up that s a the T tracked band would only be 5% of a normal peak height
on the sequencer?
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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